1. Field of the Invention
The invention relates to a method of separating and/or enriching prokaryotic DNA or of depleting said DNA from physiological liquids using a protein which specifically binds non-methylated cytidine-phosphate-guanosine dinucleotides (CpG motifs) of DNA, as well as to a kit for carrying out said method.
2. Background
Infections caused by bacteria are one of the most frequent causes of inflammatory diseases. For the prognosis of the clinical cause as well as, in particular, for timely selection of suitable therapeutic measures, early detection of the bacterial pathogens is of decisive importance.
In the detection of bacterial pathogens use is made even today, above all, of different methods of cultivating cells. However, current studies clearly show the poor suitability of culture-dependent methods for detection of pathogens (Hellebrand W., König-Bruhns C., Hass W., Studie zur Blutkulturdiagnostik im Jahr 2002, Poster Jahrestagung der Deutschen Gesellschaft Für Hygiene und Mikrobiologie, Göttingen 2004; Straube E (2003) Sepsis—microbiological diagnosis. Infection 31:284). According to these studies, it was possible to determine pathogens in only approximately 15-16% of all blood cultures examined. As a result of the disadvantages of these methods, increased efforts were made to find alternatives, especially during the past decade, simultaneously with the rapid technological development in molecular biology. First reports on the use of culture-independent methods of detecting bacterial pathogens, based on the principal of the polymerase chain reaction (PCR), date back to the early 1990s. Thus, for instance, Miller and colleagues (Miller N J Clin Microbiol. 1994 (February;32(2):393-7) were able to show that culture-independent methods are superior to the classic techniques of cultivation and microscopy for detection of mycobacterium tuberculosis. However, further molecular-biological methods based on the detection of pathogen-specific nucleic acids have gained importance (e.g. M. Grijalva et al. Heart 89 (2003) 263-268; Uyttendaele M et al. Lett Appl Microbiol. 2003;37(5):386-91; Saukkoriipi A et al. Mol Diagn. March 2003;7(1):9-15; Tzanakaki G et al. FEMS Immunol Med Microbiol. Oct. 24, 2003;39(1):31-6). In addition to the high specificity of such molecular-biological methods, the reduced time expenditure is to be mentioned as a substantial advantage over conventional culture-dependent methods. However, the sensitivity of the detection of prokaryotic DNA directly from body fluids and not from pre-treated testing material as compared to the culture of microorganisms has been much too low so far. An amount of nucleic acids of bacteria sufficient for the directed detection of pathogens from testing material, which is not pre-treated, is achieved to a limited extent only also with respect to the 16S-rRNA analysis, by means of PCR of the 16S region on the bacterial chromosome and the subsequent sequence analysis of the PCR fragment, because in most cases, several copies of the segment encoding 16S-rRNA are found on the chromosome. The direct specific detection of pathogens by means of 16S-rRNA analysis requires that only one species of pathogen is present in the sample to be examined. If there are different species of pathogens in the sample, specific detection by sequencing of the 16S-rRNA region is not possible, because the primers used are universal for most bacteria. Further, it is a prerequisite to the detection of pathogens by 16S-rRNA analysis that the bacteria to be detected are present in the metabolic phase and sufficiently express 16S-rRNA. This is usually not the case, in particular in patients subject to calculated antibiotic therapy. Moreover, expression of certain pathogenicity factors of bacteria does not occur at all times, although the corresponding genes are present in the bacterial genome. As a result, erroneously negative results are transmitted to the clinical physician. Thus, selective antibiotic therapy may be initiated either not at all or much too late. In such cases, the physician has to rely on his knowledge gained by experience and on general guidelines (such as those of the Paul Ehrlich Foundation) and will therefore effect a much too general antibiotic treatment. The unspecific use of antibiotics bears a number of risks, not only for the individual patient (such as unnecessary side effects in the form of renal damage etc.), but also for the entire society (e.g. the development of additional antibiotic resistances, such as MRSA (methicilline-resistant Staphylococcus aureus, etc.). Therefore, the detection of clinically meaningful pathogenicity factors and resistances of bacteria at the chromosomal level and at the plasmid level, i.e. ultimately on the DNA level, provides considerable advantages for the diagnosis of many infectious diseases but also of sepsis. This applies even more because, at this level, a distinction can also be made between pathogenic and commensal bacteria.
Most frequently, the detection of pathogen-specific nucleic acids is effected by nucleic acid amplification techniques (NAT), such as the amplification of the prokaryotic DNA by means of the polymerase chain reaction (PCR) or the ligase chain reaction (LCR), respectively. The high specificity and fast availability of the results is contrasted by the susceptibility to interference by contamination or by strongly reaction-inhibiting factors of clinical samples.
In a conventional PCR detection method, successful detection of pathogens in the blood theoretically requires at least 1 target DNA of the pathogen to be present in 10 μl of blood. This corresponds to approximately 100 targets in 1 ml of blood or 1,000 targets in 10 ml of blood, respectively. Things are different with regard to the blood culture for detection of infection pathogens. In this case, the lower detection limit is approximately 3-5 bacteria per 10 ml of blood.
This detection limit is presently not reached yet by PCR methods, not even by those which have their target sequences in the vicinity of the 16S-rRNA region on the chromosome. Although several regions encoding 16S-rRNA are located on the bacterial chromosome, in most cases 3 to 6, the prerequisite that at least one molecule of the template DNA is located in the PCR reaction mixture is not met.
Improved diagnostic safety is to be expected of PCR methods whose specific target sequences encode species-specific proteins, either in the chromosome or on plasmids of the microorganisms. The above remarks with respect to the detection limit also apply here. Especially under the action of a current antibiotic therapy, growth of the pathogens can be considerably decelerated, limited or blocked, even if the antibiotic employed ultimately does not have an optimal effect. This situation is often found especially in patients who are already receiving antibiotic treatment and in whom, therefore, no disease-causing bacteria can be grown from blood cultures or other samples (such as for example tracheal smears, broncho-alveolar lavages (BAL) etc.).
Due to insufficient sensitivity, the detection of pathogen-specific nucleic acids without an amplification step by direct detection of prokaryotic DNA (probe technique, FISH technique) is of diagnostic importance only at a sufficiently high germ count in the test material.
The essential problems of the detection of prokaryotic DNA for identification of bacterial pathogens in body fluids consist, in addition to PCR-inhibiting ingredients in the test material, mainly in the low concentration of prokaryotic DNA and the resulting excess of eukaryotic DNA versus prokaryotic DNA. In this connection, in particular, competitive processes in DNA analysis as well as the quantity of prokaryotic DNA can be regarded as a hindrance to qualitative and quantitative detection of pathogens.
The usual methods of DNA isolation enrich the total DNA of a body fluid so that the ratio of host DNA to microbial DNA may be between 1:10−6 and 1:10−8. This difference makes the difficulty in detecting microbial DNA in body fluids quite easy to understand.
Prokaryotic DNA differs from eukaryotic DNA, for example, by the presence of non-methylated CpG motifs (Hartmann G et al., Deutsches Ärzteblatt, Jg. 98/15:A981-A985 (2001). In the prokaryotic DNA, 16 times more CpG motifs are present than in eukaryotic DNA, which contains such motifs only temporarily, for example in cancer cells or promoter regions. These motifs are not methylated in prokaryotic DNA, whereas the majority of them are methylated in eukaryotic DNA, which further augments their distinctiveness. Non-methylated CpG motifs are non-methylated desoxycytidylate-desoxyguanylate-dinucleotides within the prokaryotic genome or within fragments thereof.
It is further known that diagnostic statements for cancers can be derived from different methylation patterns within the human DNA (Epigenetics in Cancer Prevention: Early Detection and Risk Assessment (Annals of the New York Academy of Sciences, Vol 983) Editor: Mukesh Verma ISBN 1-57331-431-5). Methylated and non-methylated cytosines in the genome allow tissue-specific but also disease-specific patterns to be identified. The specific methylation patterns of a disease allow, on the one hand, diagnosis at a very early point in time and, on the other hand, molecular classification of a disease and the likely response of a patient to a certain treatment. For detailed information on this, see, for example, Beck S, Olek A, Walter J.: From genomics to epigenomics: a loftier view of life.”, Nature Biotechnology 1999 Dec;17(12):1144, on the homepage of Epigenomics AG , or WO 200467775.
Cross at el. showed that it is possible to separate differently methylated genomic human DNA by binding the methylated CpG motifs to a protein (Cross S H, Chariton J A, Nan X, Bird A P, Purification of CpG islands using a methylated DNA binding column, Nat Genet. March 1994;6(3):236-44). Thus, this method serves to bind DNA containing methylated CpG motifs. Sufficient isolation of non-methylated and methylated DNA is not possible for technical reasons, because the protein used also weakly binds non-methylated DNA. It is also not possible with these methods to enrich non-methylated DNA, because the capacity of the protein used is not sufficient to separate non-methylated DNA to a sufficient extent in the case of a high excess of methylated DNA. Further, due to the binding of the methylated DNA, the initial volume in which the non-methylated DNA is present, remains unchanged so that no enrichment is achieved.
Thus it would be desirable to separate non-methylated DNA from methylated DNA and to be able to enrich non-methylated DNA so as to separate prokaryotic DNA from eukaryotic DNA or differently methylated human DNA, respectively, from each other. In addition, it would be desirable and of great interest in terms of health economics if the isolation and enrichment of non-methylated DNA could also be obtained from a mixture (for example, full blood) which is characterized by a great excess of methylated DNA.
It is known from Voo et al. that human CpG-binding protein (hCGBP) is capable of binding non-methylated CpG motifs. This publication describes the transcription-activating factor hCGBP which has been shown to play a role in the regulation of gene expression in CpG motifs.
EP 02020904 shows a method which enables isolation and enrichment of prokaryotic DNA from a mixture of prokaryotic and eukaryotic DNA by binding the prokaryotic DNA to a protein which specifically binds non-methylated DNA.